Effects of superparamagnetic iron oxide nanoparticles (SPIONS) testicular injection on Leydig cell function and sperm production in a murine model.

In the domain of medical advancement, nanotechnology plays a pivotal role, especially in the synthesis of biocompatible materials for therapeutic use. Superparamagnetic Iron Oxide Nanoparticles (SPIONs), known for their magnetic properties and low toxicity, stand at the forefront of this innovation. This study explored the reproductive toxicological effects of Sodium Citrate-functionalized SPIONs (Cit_SPIONs) in adult male mice, an area of research that holds significant potential yet remains largely unknown. Our findings reveal that Cit_SPIONs induce notable morphological changes in interstitial cells and the seminiferous epithelium when introduced via intratesticular injection. This observation is critical in understanding the interactions of nanomaterials within reproductive biological systems. A striking feature of this study is the rapid localization of Cit_SPIONs in Leydig cells post-injection, a factor that appears to be closely linked with the observed decrease in steroidogenic activity and testosterone levels. This data suggests a possible application in developing nanostructured therapies targeting androgen-related processes. Over 56 days, these nanoparticles exhibited remarkable biological distribution in testis parenchyma, infiltrating various cells within the tubular and intertubular compartments. While the duration of spermatogenesis remained unchanged, there were many Tunel-positive germ cells, a notable reduction in daily sperm production, and reduced progressive sperm motility in the treated group. These insights not only shed light on the intricate mechanisms of Cit_SPIONs interaction with the male reproductive system but also highlight the potential of nanotechnology in developing advanced biomedical applications.

Detection of carbon nanotubes in bovine raw milk through Fourier transform Raman spectroscopy.

The potential use of carbon-based methodologies for drug delivery and reproductive biology in cows raises concerns about residues in milk and food safety. This study aimed to assess the potential of Fourier transform Raman spectroscopy and discriminant analysis using partial least squares (PLS-DA) to detect functionalized multiwalled carbon nanotubes (MWCNT) in bovine raw milk. Oxidized MWCNT were diluted in milk at different concentrations from 25.00 to 0.01 µg/mL. Raman spectroscopy measurements and PLS-DA were performed to identify low concentrations of MWCNT in milk samples. The PLS-DA model was characterized by the analysis of the variable importance in projection (VIP) scores. All the training samples were correctly classified by the model, resulting in no false-positive or false-negative classifications. For test samples, only one false-negative result was observed, for 0.01 µg/mL MWCNT dilution. The association between Raman spectroscopy and PLS-DA was able to identify MWCNT diluted in milk samples up to 0.1 µg/mL. The PLS-DA model was built and validated using a set of test samples and spectrally interpreted based on the highest VIP scores. This allowed the identification of the vibrational modes associated with the D and G bands of MWCNT, as well as the milk bands, which were the most important variables in this analysis.

Cytocompatibility of carboxylated multi-wall carbon nanotubes in stem cells from human exfoliated deciduous teeth.

Carboxylated multi-wall carbon nanotube (MWCNT-COOH) presents unique properties due to nanoscale dimensions and permits a broad range of applications in different fields, such as bone tissue engineering and regenerative medicine. However, the cytocompatibility of MWCNT-COOH with human stem cells is poorly understood. Thus, studies elucidating how MWCNT-COOH affects human stem cell viability are essential to a safer application of nanotechnologies. Using stem cells from the human exfoliated deciduous teeth model, we have evaluated the effects of MWCNT-COOH on cell viability, oxidative cell stress, and DNA integrity. Results demonstrated that despite the decreased metabolism of mitochondria, MWCNT-COOH had no toxicity against stem cells. Cells maintained viability after MWCNT-COOH exposure. MWCNT-COOH did not alter the superoxide dismutase activity and did not cause genotoxic effects. The present findings are relevant to the potential application of MWCNT-COOH in the tissue engineering and regenerative medicine fields.

Monte Carlo simulation and dosimetry measurements of an experimental approach for in vitro HDR brachytherapy irradiation.

Irradiation of tumor cell lines is a useful way to investigate the effects of ionizing radiation on biological molecules. We designed an easy and reproducible approach for in vitro experimental high dose rate brachytherapy, which was simulated by a Monte Carlo code and dosimetrically characterized by experimental methods to evaluate the correspondence between planned doses and doses absorbed by the cells. This approach is an acrylic platform containing T25 tissue culture flasks and multiwell tissue culture plates. It allows nine parallel needles carrying an 192Ir source to irradiate the adherent cells. The whole system composed of the acrylic platform, tissue culture flasks and 192Ir source tracking was simulated by the Monte Carlo N-Particle transport code (MCNPX). Dosimetric measurements were taken by well ionization chamber and radiochromic films. There was a slight difference, averaging from 2% to 7%, between the MCNPX results and film dosimetry results regarding uniform radiation created by the source arrangement. The results showed different values for planned and measured doses in each cell culture plate, which was attributed to the non-equivalent water material used and to the lack of full scattering coming from the top of the platform. This last contribution was different for each tissue culture plate and an individual dose correction factor was calculated. The dose correction factor must be applied to match the planned dose and the actual doses absorbed by the cells. The designed approach is an efficient tool for in vitro brachytherapy experiments for most commercial cell culture plates.

Gold nanoparticles enhance fluorescence signals by flow cytometry at low antibody concentrations.

Flow cytometry is a universally applied technique in many biological and clinical assays to evaluate cells, bacteria, parasites, and particles at a micrometre scale. More advanced flow cytometers can detect small molecules down to the nanometre scale that may identify intracellular nanostructures. Advancements in the field of nanobiotechnology have led to techniques that allow the study of cellular behaviour after exposure to nanomaterials, particularly, metal nanoparticles. The optical properties of gold nanoparticles regarding surface plasmon resonance (SPR) are established to increase the fluorescence quantum yields of several dyes working as optical antennas, enabling the enhancement of light emission in fluorescent emitters. In this work we constructed a nanoprobe using gold nanoparticles coated with primary antibody Cetuximab. Then, we investigated whether this nanoprobe labelled with secondary fluorescent antibody Alexa Fluor 488, at low concentrations, could promote fluorescent signal enhancement, associated with SPR, and detected by the flow cytometry technique. Our results showed an enhanced fluorescent signal likely due to the proximity between the extinction coefficient of gold nanoparticles and the emission peak of Alexa Fluor 488, at exceptionally low concentrations, occurring within a high level of specificity. Moreover, the nanoprobe did not alter the cellular viability suggesting gold nanoparticles as a feasible approach for cell labelling using low concentrations of secondary antibodies for routine flow cytometry applications.

Essential oils of Taxandria fragrans and Melaleuca alternifolia have effective antidermatophytic activities in vitro and in vivo that are antagonised by ketoconazole and potentiated in gold nanospheres.

The investigation of the effects of three essential oils (EOs) from Taxandria fragrans (FRA), Melaleuca alternifolia (TTO) and Boswellia serrata (IF), alone and combined with ketoconazole (KTZ), and their functionalised gold nanoparticles (AuNP) against Trichophyton interdigitale both in vitro and in vivo indicated that EOs presented activity against T. interdigitale. The combination of EOs and KTZ was antagonistic. FRA, TTO, gold nanoparticles capped with T. fragrans (AuNPFRA) and gold nanoparticles capped with M. alternifolia (AuNPTTO) presented antidermatophytic activity in vivo, with the capacity to reduce fungal burden and to preserve tissue architecture; however, combination treatment with KTZ increased fungal burden and caused tissue damage. The combination of EO with KTZ exhibited antagonistic activity and was histologically harmful. In contrast, FRA, TTO, AuNPFRA and AuNPTTO are promising treatments for dermatophytosis.

Antileishmanial activity of fullerol and its liposomal formulation in experimental models of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2 mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2 mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1ß cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.

Nanosensors based on LSPR are able to serologically differentiate dengue from Zika infections.

The Flaviviridae virus family was named after the Yellow-fever virus, and the latin term flavi means "of golden color". Dengue, caused by Dengue virus (DENV), is one of the most important infectious diseases worldwide. A sensitive and differential diagnosis is crucial for patient management, especially due to the occurrence of serological cross-reactivity to other co-circulating flaviviruses. This became particularly important with the emergence of Zika virus (ZIKV) in areas were DENV seroprevalence was already high. We developed a sensitive and specific diagnostic test based on gold nanorods (GNR) functionalized with DENV proteins as nanosensors. These were able to detect as little as one picogram of anti-DENV monoclonal antibodies and highly diluted DENV-positive human sera. The nanosensors could differentiate DENV-positive sera from other flavivirus-infected patients, including ZIKV, and were even able to distinguish which DENV serotype infected individual patients. Readouts are obtained in ELISA-plate spectrophotometers without the need of specific devices.

Biotechnological approach to induce human fibroblast apoptosis using superparamagnetic iron oxide nanoparticles.

Cancer-Associated Fibroblasts (CAFs) contribute to tumour progression and have received significant attention as a therapeutic target. These cells produce growth factors, cytokines and chemokines, stimulating cancer cell proliferation and inhibiting their apoptosis. Recent advances in drug delivery have demonstrated a significant promise of iron oxide nanoparticles in clinics as theranostic agents, mainly due to their magnetic properties. Here, we designed superparamagnetic iron oxide nanoparticles (SPIONs) to induce apoptosis of human fibroblasts. SPIONs were synthesized via co-precipitation method and coated with sodium citrate (SPION_Cit). We assessed the intracellular uptake of SPIONs by human fibroblast cells, as well as their cytotoxicity and ability to induce thermal effects under the magnetic field. The efficiency and time of nanoparticle internalization were assessed by Prussian Blue staining, flow cytometry and transmission electron microscopy. SPIONs_Cit were detected in the cytoplasm of human fibroblasts 15 min after in vitro exposure, entering into cells mainly via endocytosis. Analyses through Cell Titer Blue assay, AnnexinV-fluorescein isothiocyanate (FITC) and propidium iodide (PI) cellular staining demonstrated that concentrations below 8 × 10-2 mg/mL of SPIONs_Cit did not alter cell viability of human fibroblast. Furthermore, it was also demonstrated that SPIONs_Cit associated with alternating current magnetic field were able to induce hyperthermia and human fibroblast cell death in vitro, mainly through apoptosis (83.5%), activating caspase 8 (extrinsic apoptotic via) after a short exposure period. Collectively these findings suggest that our nanoplatform is biocompatible and can be used for therapeutic purposes in human biological systems, such as inducing apoptosis of CAFs.

The physicochemical and biological characterization of a 24-month-stored nanocomplex based on gold nanoparticles conjugated with cetuximab demonstrated long-term stability, EGFR affinity and cancer cell death due to apoptosis.

Nanotechnology is one of the most promising tools for future diagnosis and therapy. Thus, we have produced gold nanoparticles coated with cetuximab at a dose-range from 5 µg up to 200 µg, and prolonged stable nanocomplexes were obtained. The nanocomplexes were characterized by UV-Vis, zeta potential, TEM, fluorometry, infrared regions, XPS and atomic absorption spectrometry. For biological characterization the A431 cell line was used. Cellular uptake, target affinity and cell death were assessed using ICP-OES, immunocytochemistry and flow cytometry, respectively. The immobilization of cetuximab on the AuNPs surfaces was confirmed. The nanocomplex with 24 months of manufacturing promoted efficient EGFR binding and induced tumour cell death due to apoptosis. Significant (p < 0.05) cell death was achieved using relatively low cetuximab concentration for AuNPs coating compared to the antibody alone. Therefore, our results provided robust physicochemical and biological characterization data corroborating the cetuximab-bioconjugate AuNPs as a feasible nanocomplex for biomedical applications.

Effect of Multi-walled Carbon Nanotubes on Metabolism and Morphology of Filamentous Green Microalgae.

Multi-walled carbon nanotubes (MWCNTs) have potential applications in the industrial, agricultural, pharmaceutical, medical, and environmental remediation fields. However, many uncertainties exist regarding the environmental implications of engineered nanomaterials. This study examined the effect of the MWCNTs on metabolic status and morphology of filamentous green microalgae Klebsormidium flaccidum. Appropriate concentrations of MWCNT (1, 50, and 100 µg mL-1) were added to a microalgal culture in the exponential growth phase and incubated for 24, 48, 72, and 96 h. Exposure to MWCNT led to reductions in algal growth after 48 h and decreased on cell viability for all experimental endpoints except for 1 µg mL-1 at 24 h and 100 µg mL-1 after 72 h. At 100 µg mL-1, MWCNTs induced reactive oxygen species (ROS) production and had an effect on intracellular adenosine triphosphate (ATP) content depending on concentration and time. No photosynthetic activity variation was observed. Observations by scanning transmission electron microscopy showed cell damage. In conclusion, we have demonstrated that exposure to MWCNTs affects cell metabolism and microalgal cell morphology. To our best knowledge, this is the first case in which MWCNTs exhibit adverse effects on filamentous green microalgae K. flaccidum. These results contribute to elucidate the mechanism of MWCNT nanotoxicity in the bioindicator organism of terrestrial and freshwater habitats.

HDR brachytherapy decreases proliferation rate and cellular progression of a radioresistant human squamous cell carcinoma in vitro.

PURPOSE: To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters. MATERIALS AND METHODS: An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method. RESULTS: Significant decreasing cellular proliferation rate (p < 0.05) as well as reduced survival fraction (p < 0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G2/M phase (32.3 ± 1.5%) in the irradiated group as compared with untreated cells (8.22 ± 1.2%) was detected. Also, a significant (p < 0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found. CONCLUSION: HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G2/M cell cycle arrest.

Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice.

BACKGROUND: Dengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue. METHODS: The MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model. RESULTS: Immunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E. CONCLUSIONS: Despite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine's phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.

Encapsulation of nor-ß-lapachone into poly(d,l)-lactide-co-glycolide (PLGA) microcapsules: full characterization, computational details and cytotoxic activity against human cancer cell lines.

In this work, we characterize nor-ß-lapachone-loaded (NßL-loaded) microcapsules prepared using an emulsification/solvent extraction technique. Features such as surface morphology, particle size distribution, zeta potential, optical absorption, Raman and Fourier transform infrared spectra, thermal analysis data, drug encapsulation efficiency, drug release kinetics and in vitro cytotoxicity were studied. Spherical microcapsules with a size of 1.03 ± 0.46 µm were produced with an encapsulation efficiency of approximately 19%. Quantum DFT calculations were also performed to estimate typical interaction energies between a single nor-ß-lapachone molecule and the surface of the microparticles. The NßL-loaded PLGA microcapsules exhibited a pronounced initial burst release. After the in vitro treatment with NßL-loaded microcapsules, a clear phagocytosis of the spheres was observed in a few minutes. The cytotoxic activity against a set of cancer cell lines was investigated.

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.

A novel 3D bone-mimetic scaffold composed of collagen/MTA/MWCNT modulates cell migration and osteogenesis.

AIMS: This study characterized a three-dimensional (3D) biocomposite scaffolds produced using type I collagen, mineral trioxide aggregate (MTA) and multi-walled carbon nanotubes (MWCNT) to be used in bone tissue regeneration. MAIN METHODS: The scaffolds were analyzed via scanning (SEM) and transmission (TEM) electron microscopy, as well as the viability and migration of osteoblasts and mineralization of the scaffolds. KEY FINDINGS: SEM and TEM analyses showed that MTA and MWCNT were distributed as both large agglomerates entrapped within the collagen network and as smaller accumulations or individual molecules dispersed throughout the scaffold. Ultrastructural analysis revealed that osteoblastic MC3T3-E1 cells grown in the biocomposite endocytosed MWCNT, which were localized in the cytoplasm and in vesicles. Analysis of cells grown in the 3D scaffolds demonstrated that >95% of the cells remained viable in all tested combinations and concentrations of the biocomposite. MC3T3-E1 osteoblasts migrated into scaffolds formed with concentrations of type I collagen between 1.75 and 3.0mg/mL. Cells displayed increased migration into scaffolds formed with collagen and a range of low to high concentrations of MTA. In contrast, the presence of MWCNT in the biocomposite had a slight negative effect on migration. Collagen gels containing specific concentrations of MTA, or MWCNT, or combinations of MTA/MWCNT, caused an increase in mineralization of scaffolds. SIGNIFICANCE: Scaffolds composed of defined concentrations of type I collagen, MTA and MWCNT are biocompatible, promote migration and mineralization of osteoblasts, and hence may be useful as bone tissue mimetics.

Controlled Release of Nor-ß-lapachone by PLGA Microparticles: A Strategy for Improving Cytotoxicity against Prostate Cancer Cells.

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-ß-lapachone (NßL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 µm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NßL on PLGA. Finally, the cytotoxic activity of NßL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

Biocompatibility assessment of fibrous nanomaterials in mammalian embryos.

Currently there is a growing interest in the use of nanotechnology in reproductive medicine and reproductive biology. However, their toxic effects on mammalian embryos remain poorly understood. In this work, we evaluate the biocompatibility of two fibrous nanomaterials (NMs): cotton cellulose nanofibers (CNF) and carboxylated multiwalled carbon nanotubes (MWCNT-COOH), by performing an investigation of the embryonic development, gene expression (biomarkers focused on cell stress, apoptosis and totipotency) and in situ apoptosis in bovine embryos. Exposure to NMs did not interfere in preimplantation development or in the incidence of apoptosis in the bovine embryo, but they did affect the gene expression. The results presented are important for an understanding of the toxicity of cotton CNF and MWCNT-COOH on mammalian embryos. To our knowledge, we report the first evaluation of biocompatibility between these NMs on preimplantation embryos, which may open a new window for reproductive biomedical applications.

Graphene-based nanomaterials: biological and medical applications and toxicity.

Graphene and its derivatives, due to a wide range of unique properties that they possess, can be used as starting material for the synthesis of useful nanocomplexes for innovative therapeutic strategies and biodiagnostics. Here, we summarize the latest progress in graphene and its derivatives and their potential applications for drug delivery, gene delivery, biosensor and tissue engineering. A simple comparison with carbon nanotubes uses in biomedicine is also presented. We also discuss their in vitro and in vivo toxicity and biocompatibility in three different life kingdoms (bacterial, mammalian and plant cells). All aspects of how graphene is internalized after in vivo administration or in vitro cell exposure were brought about, and explain how blood-brain barrier can be overlapped by graphene nanomaterials.

Physiological changes of the lichen Parmotrema tinctorum as result of carbon nanotubes exposition.

Carbon nanotubes (CNT) is one of the more abundant nanomaterial produced in the world. Therefore, it is desirable to access its effects in all environment compartments, in order to mitigate environmental distress. This study aims to verify the potential use of lichens - classical atmospheric pollution indicators - as biomonitors of carbon nanotubes aerosols. To examine cause-effect relationships, preserving environmental microclimatic parameters, the lichen Parmotrema tinctorum (Nyl.) Hale was transplanted to open top chambers where aerosols of CNT were daily added. Physiological parameters such as cell viability, photosynthetic efficiency, cell permeability as well as nanoparticle internalization were assessed. Carbon nanotubes exposure led to reduction on the cell viability of P. tinctorum. The treatment with 100µg/mL of MWCNT-COOH resulted in intracellular ion leakage, probably due to changes in membrane permeability. No alterations on photosynthetic efficiency were detected. Carbon nanotubes entrapment and internalization into the lichen thallus were observed. Short term exposition of CNT produced measurable physiological changes in P. tinctorum lichen. This suggests the possibility of use of lichens as models to assess the environmental impact (air related) of engineered nanomaterials.